Animals including humans contain a small number of cells in their gonads called germline stem cells (GSCs). These cells are essential for making sperm or eggs throughout the lifetime of the animal. The decision for a dividing stem cell to renew as a stem cell or to differentiate, and the regulation of early divisions in the germline are some of the most critical decisions in development. Studies of the cellular signaling for this process in the fruit fly, Drosophila melanogaster, have discovered many of the key genes involved. The importance of proper regulation of this process in all animal species would lead one to predict that these functions are highly conserved. However, several of the key GSC regulatory molecules have recently been discovered to be rapidly evolving under adaptive evolution in two closely related species of Drosophila (D. melanogaster and its sibling species D. simulans) but not in several additional closely related species. These adaptively evolving genes have accumulated a highly significant excess of amino-acid changes compared to neutrally evolving genes. Identifying the functional consequences of GSC gene evolution and the evolutionary forces driving these changes is essential for understanding how GSCs are regulated during normal development and how their misregulation can lead to infertility, germline cancers, and reproductive isolation. This proposal focuses on evaluating both the functional consequences and the evolutionary forces responsible for driving high rates of protein divergence at two key GSC regulator genes, bam and bgcn. Evolutionary patterns of GSC regulatory genes will also be examined in other Drosophila species. Numerous hypotheses have been proposed to explain the evolutionary mechanisms driving the previously discovered rapid protein evolution of other reproductive genes. Based on preliminary functional and evolutionary data, this proposal focuses on testing the hypothesis that natural selection to modulate the effects of or defend against germline endosymbionts is a key driver of adaptive evolution of bam and bgcn. These studies provide a framework with which to test for similar selectively driven functional changes in other genes controlling stem cell fate decisions in Drosophila as well as in other organisms including humans, and for understanding the evolutionary forces driving these evolutionary changes. PUBLIC HEALTH RELEVANCE: Germline stem cells (GSCs) are arguably the most fundamental cell type required for animal reproduction because these cells both maintain the germline throughout the animal's life, and produce the cells that go on to form either sperm or eggs. Remarkably, several key GSC regulatory genes are under strong natural selection to change at the protein sequence level between species. Our proposed study is aimed at identifying the functional consequences of GSC gene evolution and to test the germline endosymbiont conflict hypothesis as a key evolutionary driver of these changes.